All referenced patents and applications and publications are incorporated herein by reference in their entirety. Furthermore, where a definition or use of a term in a reference, which is incorporated by reference herein is inconsistent or contrary to the definition of that term provided herein, the definition of that term provided herein shall control.
The production of molecules by cells depends on many factors including the growth rate of the cells, the concentration of cells, the concentration of nutrients, the concentration of waste products, and the concentration of desired products. Therefore, a preferred cell culture system for developing cell culture bioprocesses will be able to independently control these factors. In addition, because many experiments may be required to optimize a cell culture bioprocess, a preferred cell culture system will also be able to perform many experiments economically.
A typical bioreactor known in the art can be operated in three modes, batch, fed-batch, and continuous. In batch mode, no additional nutrients are added to the bioreactor throughout the bioreaction. In fed-batch mode, additional nutrients are added and the volume of fluid in the bioreactor can increase throughout the bioreaction. In continuous mode, fluid is added to and removed from the bioreactor so that the volume of fluid in the bioreactor remains nominally constant.
In batch and fed-batch mode, the concentration of cells, the concentration of nutrients, and the concentration of waste and desired products all change over time. This makes batch and fed-batch processes difficult to characterize and specify for product quality control.
Continuous mode cultures come closer to the preferred cell culture system because their operation can generally maintain cell cultures in a steady state condition where the concentration of cells, the concentration of nutrients, and the concentration products remain constant. Continuous mode cultures also have the ability to control metabolic parameters such as the growth rate.
A chemostat is a type of continuous culture where the growth rate is controlled by setting the dilution rate. The dilution rate is the flow rate through the bioreactor divided by the volume of fluid in the bioreactor, both of which are held constant. There is an inflow of fresh nutrient medium and an outflow of the mixed contents of the bioreactor. The chemostat reaches a steady state condition when the cell concentration increases to the point where the number of cells removed in the outflow equals the number of cells that grow from the influx of nutrients from the inflow. Because the number of cells produced per unit time and the number of cells removed per unit time are directly proportional to the cell concentration, in steady state the growth rate equals the dilution rate. The cell growth from the influx of nutrients is determined by the inflow concentration of a limiting nutrient. Therefore, the cell concentration in a chemostat can be controlled by adjusting the concentration of the limiting nutrient.
One limitation of a chemostat is that cells cannot be set to grow near their maximum growth rate because under this condition the cell density decreases and a washout condition can occur where no cells remain in the bioreactor.
To grow cells at their maximum growth rate in continuous culture, turbidostat control is used. In a turbidostat the cell density is controlled by adjusting the dilution rate. When the cell density, as measured by the turbidity of the culture rises above the setpoint, the dilution rate is increased, and when the cell density falls below the setpoint, the dilution rate is decreased. In this mode of culture, the cell density is constant and cells can be maintained at their maximum growth rate.
With the use of a chemostat or a turbidostat a full range of growth rates can be controlled for the cell culture, and the cell density can be controlled, however product concentrations cannot be directly controlled.
In chemostat or turbidostat mode, the product concentrations are determined by the dilution rate, cell density, and cellular product production rate. The product concentrations will change until the product production rate is equal to the product removal rate. The product production rate is equal to the cellular product production rate multiplied by the cell density and the product removal rate is equal to the product concentration multiplied by the outflow rate. Therefore, for a given dilution rate, a higher product production rate will result in a higher product concentration.
The inability to independently control product concentrations is very important when the production rate of a particular product is influenced by the concentration of the product or other products. For example, if production of a protein is inhibited by the concentration of an organic acid and the organic acid production rate is relatively high, for low dilution rates, the organic acid concentration will be relatively high and inhibit the protein production. Another example is if the production of ethanol is highest when the growth rate is low, but the production of ethanol is inhibited by high concentrations of ethanol. In this case, high ethanol productivity cannot be achieved since a low growth rate implies a low dilution rate and a high ethanol concentration.
To better control product concentrations, a bioreactor can be operated in a continuous perfusion mode. In perfusion mode, there is a constant inflow of nutrients and a constant outflow, however cells are prevented from leaving the bioreactor in the outflow. In perfusion mode, the total flow rate through the bioreactor can be set arbitrarily, independent of the growth rate of the cells. As such, the product concentrations can be controlled to the extent that increasing the dilution rate of molecules decreases their concentration. However, in perfusion mode, the concentration of cells will increase until cell growth stops due to nutrient limitation since the nutrient delivery rate is still related to the flow rate. Therefore, while perfusion culture allows more control over product concentration through diluting the molecules and not the cells, a constant cell density cannot be maintained at a non-zero growth rate.
A significant problem with conventional continuous culture systems is their relatively large working volume of 0.5 liters to 10 liters. Because 10 to 100 times the working volume is typically required for an experiment, small working volumes of 100 microliters to 10 mililiters are highly desirable for continuous culture systems, especially if many experiments are conducted for process development.
In order to perform a chemostat, turbidostat, or perfusion culture, a common requirement is the ability to control the flow rate into and out of the bioreactor and to control the volume of fluid within the bioreactor. For conventionally sized bioreactors with volumes on the order of 0.5 liter to 1000 liters, conventional liquid pumps and flow meters can serve to control the fluid flow. To control the volume, many methods are available to measure the liquid volume, such as gravimetric methods or liquid level measurement methods. The liquid volume measurement can then be used to actively remove or add fluid to keep the volume constant. Alternatively, passive methods where fluid above a determined liquid level is not retained in the bioreactor can be used.
However, for small volume bioreactors, with working volumes approximately 100 micro liters to 10 mililiters, fluid flow measurement and control and fluid volume measurement and control are difficult to implement. Such small volume bioreactors are very desirable for continuous culture experiments, however, due to the dramatic reduction in total fluid used in each processing run.
Thus, there remains a considerable need for apparatus and methods that can provide independent control over growth rate, cell density, and product concentrations in continuous culture bioreactors, particularly for small scale bioreactors.